Dynamic changes in circulating free rna of neural tumors

ABSTRACT

Circulating free RNA (cfRNA) is used for monitoring status and/or treatment response for neural tumors, and especially glioma, glioblastoma, and neuroblastoma. Particularly preferred cfRNAs include those that encode a marker that is specific to a neural tumor, but also markers that are specific to DNA repair status and/or immune status.

This application claims priority to our co-pending US provisionalapplication having the Ser. No. 62/547,047, filed Aug. 17, 2017, whichis incorporated in its entirety herein.

FIELD OF THE INVENTION

The field of the invention is methods of monitoring progression,pseudo-progression, and treatment response of neural tumors, especiallyas it relates to brain tumors such as glioblastoma, glioma andneuroblastoma using circulating free RNA (cfRNA).

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

Neural tumors, especially brain tumors, present a unique challenge toclinicians and researchers as the tumor is often protected within theblood-brain barrier and as such is difficult to reach withpharmaceutical agents. Likewise, monitoring of treatment and tumorprogression without direct access to the tumor is typically performedusing various imaging methods (e.g., contrast enhanced MRI and CT, ormagnetic resonance spectroscopy) as most analytes remain behind theblood-brain barrier. Unfortunately, even imaging technologies are atleast to some degree dependent on the function of the blood-brainbarrier, which is often affected by various drugs. Worse yet, inmalignant gliomas, imaging can generally not reliably distinguishtreatment-related changes such as radiation necrosis from actual tumorgrowth. As a result, it is often impossible to make informed decisionson the effect of a specific treatment. Indeed, pseudo-progression isoften observed after radiation of high-grade gliomas that can lead apractitioner to premature discontinuation of adjuvant chemotherapy, andeven repeat surgery. Conversely, the mirror image of this problem is apseudo-response that can be seen when edema and blood-brain barrierpermeability decrease without an actual reduction in tumor burden.

While it should at least conceptually be difficult for circulating tumorcells to penetrate the blood-brain barrier, indirect evidence suggestedthat CTCs are indeed present in patients with malignant gliomas.Although metastatic glioblastoma is rarely observed in clinical practice(0.4-0.5% of glioblastoma cases) it can occur, with numerous cases ofmetastatic glioblastoma reported in the literature. In addition, anumber of instances of GBM transmission have been reported in patientswho received organ transplants from donors with GBM, and it has beenestimated that between 12.5 and 25% of donors with GBM might transmitthe tumor (Neuro Oncol. 2004; 6:259-263). These cases provide directevidence that GBM tumor cells were present in donated organs at the timeof transplant surgery and the cells must have migrated out of the brainvia the bloodstream. Unlike in other solid tumors, however, CTCs havenot yet been successfully detected in patients with gliomas.

Circulating proteins have been used as tumor markers in a variety ofcancers. Many proteins that are possible biomarkers for glioma wereinitially identified as markers of traumatic or hypoxic brain injury. Aprototype glial-specific marker is glial fibrillary acidic protein(GFAP). Serum levels of GFAP increase after stroke and traumatic braininjury and appear to also be increased in the blood of patients withhigh-grade gliomas. A study of patients undergoing surgery for suspectedglioma, however demonstrated that serum GFAP increases after resectionregardless of tumor grade, suggesting that increased serum GFAP is amarker of brain injury and not a specific marker of tumor. Still furtherprotein and nucleic acid markers useful for biopsy materials aredescribed in EP 2260303, U.S. Pat. No. 7,754,426, and US 2003/0108915.

It is also well known that cancers shed DNA into the bloodstream, andcirculating tumor DNA (ctDNA) has been demonstrated in a number of solidtumors, including colorectal cancer and breast cancer. ctDNA can be ahighly sensitive and specific biomarker (see e.g., Nat Med. 2008;14:985-990). Several pilot studies have shown that circulating tumor DNAcan be detected in the blood of patients with malignant gliomas, and amore recent study showed that mutated IDH-1 DNA can be detected in theplasma of patients with IDH1-positive gliomas, and that there appearedto be a relationship between higher rates of IDH-1 DNA detectability andblood-brain barrier disruption (Neurology. 2012; 79:1693-1698).Moreover, one study analyzed methylation of O⁶-methyl-guanine-DNAmethyltransferase (MGMT), p16, DAPK and RASSF1A in serum and tumor of 28patients with glioblastoma and showed sensitivity and specificity ofover 75% for each of these methylated genes using a methylation-specific(MSP) PCR-based assay (Clin Cancer Res. 2003; 9:1461-1468). Whileconceptually promising, analysis of ctDNA remains difficult with respectto at least sensitivity and specificity of such tests.

Further known plasma-based biomarkers include tumor-derived microRNAs(miRNA). In a study of blood from 20 patients with GBM and 20age-matched controls, 1158 miRNAs were tested. Notably, two miRNAs werefound to be significantly altered in GBM patients, miR-128 (upregulated)and miR-342-3p (downregulated) (J Neurochem. 2011; 118:449-457).However, direct association of miRNAs with tumor growth and status isoften problematic.

Tumor-derived nucleic acids (and other cellular molecules) can also befound in circulating microvesicles that are directly released fromglioblastoma cells. Microvesicles can carry specific genetic informationfrom the tumor into the periphery. For example, specific EGFRvIII couldbe detected in serum microvesicles from 7 out of 25 patients that werecollected on the day of surgery (sensitivity 50%, specificity 87%,compared with EGFRvIII in tissue) (Nat Cell Biol. 2008; 10:1470-1476).However, the dynamic range using these markers is unclear, and isolationprotocols and with that test results vary significantly.

Therefore, even though numerous methods of diagnostic tests forneural/brain tumors are known in the art, all or almost all of themsuffer from various disadvantages. Consequently, there remains a needfor improved systems and methods to monitor neural/brain tumors,particularly in a non-invasive manner.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to various compositions,systems, and methods of measuring circulating free RNA (cfRNA) tomonitor status and/or treatment of neural tumors, and especiallymeasuring cfRNA for cancer markers (e.g., PD-L1) in glioblastoma,glioma, and neuroblastoma. Notably, the inventors discovered that suchcfRNA can be used as a diagnostic marker with high sensitivity,specificity, and large dynamic range to ascertain the status and tomonitor treatment of a tumor even where the tumor is protected by theblood-brain barrier.

In one aspect of the inventive subject matter, the inventors contemplatea method of monitoring status or treatment of a neural cancer of apatient in which a sample of a bodily fluid of the patient is firstobtained. Then, at least one cfRNA is quantified in the bodily fluid ofthe patient, wherein the cfRNA is specific to at least one of the neuralcancer, a DNA repair status, and an immune status. Where desired, thebarrier property of the blood-brain barrier in the patient may bemodulated (e.g., to increase cfRNA in the circulation).

Most typically, the bodily fluid is whole blood, plasma, or serum, andthe neural cancer is a glioma, a glioblastoma, or a neuroblastoma. Whilenot limiting to the inventive subject matter, the cfRNA is from a neuraltumor cell and especially contemplated cfRNAs encode at least a portionof MGMT, IDH1, EGFR, p53, PI3K, Rb, RAF, CD133, CD15, A2B5, nestin,ALDH1, ELTD-1, VEGF, PTEN, cytochrome c oxidase, MYCN, CD44, TrkA, LDH,and/or NSE. Of course, it should be noted that suitable cfRNAs includefull length versions, splice variants and all known and idiosyncraticmutations thereof.

Additionally, or alternatively, the cfRNA may also be associated withDNA repair (e.g., with base excision repair, mismatch repair, nucleotideexcision repair, homologous recombination, and/or non-homologousend-joining), and/or with an immune status of the patient (e.g., cfRNAencodes at least a portion of a PD-L1 gene). Thus, it should beappreciated that contemplated methods may also include a step ofmeasuring at least one additional cfRNA in the bodily fluid of thepatient (e.g., housekeeping or reference gene). As appropriate, one ormore ratios may be calculated where more than one cfRNA is measured(e.g., PD-L1 to beta actin).

In another aspect of the inventive subject matter, the inventorscontemplate a method of determining a prognosis of a neural cancer of apatient from the sample of a bodily fluid of the patient. In thismethod, a first sample of a bodily fluid of the patient is obtained, anda plurality of changes of one or more cfRNA in the first sample isdetected. Most preferably, wherein the bodily fluid is at least one ofwhole blood, plasma, serum, and cerebrospinal fluid and/or the cfRNA isspecific to at least one of the neural cancer, a DNA repair status, andan immune status. Then, the prognosis of the neural cancer can bedetermined based on at least one of an interrelationship among theplurality of changes and a predetermined threshold of at least one ofthe plurality of changes. Optionally, the interrelationship among theplurality of changes is measured in a sliding scale. In someembodiments, the method can further include a step of calculating ascore for the plurality of changes, and comparing the score with thepredetermined threshold.

Most typically, the plurality of changes is selected from a groupconsisting of a mutation, differential expression of splicing variants,an overexpression, an underexpression, a maturation. In one embodiment,the plurality of changes comprises a mutation in a first cfRNA and anoverexpression of a second cfRNA. In such embodiment, the first andsecond cfRNAs can be derived from two distinct genes. In anotherembodiment, the plurality of changes comprises expression levels of afirst cfRNA and a second cfRNA. In such embodiment, the first and secondcfRNAs can be derived from two distinct genes in a same signalingpathway. The first and second cfRNAs are derived from a same type ofcell or from different types of cells.

Optionally, the method may also include steps of obtaining a secondsample of a bodily fluid of the patient in a different time point thanthe first sample, detecting a plurality of changes of the one or morecfRNAs in the second sample, and determining the status of an neuralcancer by comparing the plurality of changes of the first and secondsamples. In such embodiment, it is preferred that the plurality ofchanges of the first and second sample include changes of at least onecommon cfRNA. Preferably, the different time point can be apost-treatment time point. In such embodiment, the method may furthercomprise a step of determining an effectiveness of a treatment based onthe plurality of changes of the first and second sample.

In some embodiments, the method may further include a step of modulatinga barrier property of the blood-brain barrier in the patient. Further,the method may also include a step of predicting a likelihood of successof a treatment regimen based on the prognosis and/or administering atreatment regimen based on the prognosis.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments.

DETAILED DESCRIPTION

There is an unmet need to evaluate tumor status and treatment responsefor various brain tumors, and especially glioma, glioblastoma, andneuroblastoma by other means than radiology tests or biopsy from aprimary or residual tumor or new metastasis. Advantageously, cfRNA(cell-free RNA that is derived from tumor cells and found as circulatingRNA in biological fluids) can be extracted from plasma of cancerpatients in less invasive and less complicated manner, and has beenemerged as a substitute for a tissue biopsy to obtain information neededfor cancer diagnosis. Further, the inventors have now discovered thatcfRNA can be used to evaluate tumor status and treatment response forvarious brain tumors by quantifying numerous marker cfRNAs of braintumors and their dynamic changes in gene expression.

Thus, in one especially preferred aspect of the inventive subjectmatter, the inventors contemplate a method of monitoring status ortreatment of a neural cancer of a patient using marker cfRNAs obtainedfrom the patient's bodily fluid. Most typically, suitable tissue sourcesinclude whole blood, which is preferably provided as plasma or serum.Alternatively, it should be noted that various other bodily fluids arealso deemed appropriate so long as cell free nucleic acids, preferablythose derived from neural tumors in the central nervous system includingneural tumor cells or immune cells in the neural tumor, are present insuch fluids. Appropriate fluids include ascites fluid, cerebrospinalfluid, urine, saliva, etc., which may be fresh or preserved/frozen.Moreover, it is contemplated that where desirable the blood-brainbarrier may be modulated with one or more pharmaceutical agents or othertreatment modalities (e.g., hyperosmotic agents, focused ultrasound) toincrease diffusion or other transport of cfRNA across the blood-brainbarrier. Further considerations, suitable cfRNAs, and methods aredescribed in a US provisional patent application having Ser. No.62/522,615, filed 20 Jun. 17, to which the international patentapplication No. PCT/US18/38198 claims priority, a US provisional patentapplication having Ser. No. 62/513,706, filed 1 Jun. 17, to which theinternational patent application No. PCT/US18/31764 claims priority, aU.S. provisional patent application 62/500,497, filed 3 May 17, to whichthe international patent application No. PCT/US18/30472 claims priority,and a U.S. provisional patent application 62/522,509, filed 20 Jun. 17,to which the international patent application No. PCT/US18/22747 claimspriority.

Any suitable methods of obtaining cell free nucleic acid including cellfree RNA (cfRNA) and/or cell free DNA (cfDNA) from the patient arecontemplated. In one especially preferred embodiment, specimens wereaccepted as 10 ml of whole blood drawn into cell-free RNA BCT® tubes orcell-free DNA BCT® tubes containing RNA or DNA stabilizers,respectively. Advantageously, cfRNA is stable in whole blood in thecell-free RNA BCT tubes for seven days while cfDNA is stable in wholeblood in the cell-free DNA BCT Tubes for fourteen days, allowing timefor shipping of patient samples from world-wide locations without thedegradation of cfRNA or cfDNA. Moreover, it is generally preferred thatthe cfRNA is isolated using RNA stabilization agents that will not orsubstantially not (e.g., equal or less than 1%, or equal or less than0.1%, or equal or less than 0.01%, or equal or less than 0.001%) lyseblood cells. Viewed from a different perspective, the RNA stabilizationreagents will not lead to a substantial increase (e.g., increase intotal RNA no more than 10%, or no more than 5%, or no more than 2%, orno more than 1%) in RNA quantities in serum or plasma after the reagentsare combined with blood. Likewise, these reagents will also preservephysical integrity of the cells in the blood to reduce or even eliminaterelease of cellular RNA found in blood cell. Such preservation may be inform of collected blood that may or may not have been separated. In lesspreferred aspects, contemplated reagents will stabilize cfDNA and/orcfRNA in a collected tissue other than blood for at 2 days, morepreferably at least 5 days, and most preferably at least 7 days. Ofcourse, it should be recognized that numerous other collectionmodalities are also deemed appropriate, and that the cfRNA and/or cfDNAcan be at least partially purified or adsorbed to a solid phase to soincrease stability prior to further processing.

As will be readily appreciated, fractionation of plasma and extractionof cfDNA and cfRNA can be done in numerous manners. In one exemplarypreferred aspect, whole blood in 10 mL tubes is centrifuged tofractionate plasma at 1600 rcf for 20 minutes. The so obtained plasma isthen separated and centrifuged at 16,000 rcf for 10 minutes to removecell debris. Of course, various alternative centrifugal protocols arealso deemed suitable so long as the centrifugation will not lead tosubstantial cell lysis (e.g., lysis of no more than 1%, or no more than0.1%, or no more than 0.01%, or no more than 0.001% of all cells). cfDNAand cfRNA are extracted from 2 mL of plasma using Qiagen reagents. Theextraction protocol was designed to remove potential contaminating bloodcells, other impurities, and maintain stability of the nucleic acidsduring the extraction. All nucleic acids were kept in bar-coded matrixstorage tubes, with DNA stored at −4° C. and RNA stored at −80° C. orreverse-transcribed to cDNA that is then stored at −4° C. Notably, soisolated cfRNA can be frozen prior to further processing.

Various types of omics data on such obtained patient's cfDNA and/orcfRNA can be generated and changes in the omics data can be determinedby comparing the omics data of a healthy individual or the omics data ofthe patient that is generated at a different time point. As used herein,omics data includes information related to genomics (e.g., DNA sequenceinformation, etc.), and transcriptomics (e.g., RNA sequence information,RNA expression level, splicing variants, etc.). Thus, changes in theomics data may include a change in a DNA or a RNA sequence compared to ahealthy individual or the patient's own tissue (e.g., DNA obtained fromhealthy tissue such as muscle, liver, skin, etc., or cfDNA or cfRNAobtained from different time point), which comprises missense mutation,nonsense mutation, deletion, insertion, duplication, frameshiftmutation, repeat expansion, length of poly A tail, etc.). Changes in theomics data may also include a change in expression levels (e.g.,upregulation, downregulation, etc.) of RNAs (cellular mRNA or regulatoryRNA (e.g., miRNA, etc.)), a change in types or ratios of splicingvariants of an mRNA of a gene, or maturation status of the mRNA (e.g.,splicing status, length of poly A tail, etc.).

There are numerous methods of genomics and/or transcriptomic analysisknown in the art, and all of the known methods are deemed suitable foruse herein (e.g., next-generation sequencing (NGS), RNAseq, RNAhybridization arrays, qPCR, etc.). For example, for transcriptomicsanalysis, preferred materials include mRNA and primary transcripts(hnRNA), and RNA sequence information may be obtained from reversetranscribed polyA⁺-RNA, which is in turn obtained from a tumor sampleand a matched normal (healthy) sample of the same patient. Likewise, itshould be noted that while polyA⁺-RNA is typically preferred as arepresentation of the transcriptome, other forms of RNA (hn-RNA,non-polyadenylated RNA, siRNA, miRNA, etc.) are also deemed suitable foruse herein. Preferred methods include quantitative RNA (hnRNA or mRNA)analysis and/or quantitative proteomics analysis, especially includingRNAseq. In other aspects, RNA quantification and sequencing is performedusing RNA-seq, qPCR and/or rtPCR based methods, although variousalternative methods (e.g., solid phase hybridization-based methods) arealso deemed suitable.

Quantification of cfRNA can be performed in numerous manners, however,expression of analytes is preferably measured by quantitative real-timePCR of cf-cDNA using primers specific for each gene. For example,amplification can be performed using an assay in a 10 μL reaction mixcontaining 2 μL cDNA, primers, and probe. β-actin can be used as aninternal control for the input level of cf-cDNA. A standard curve ofsamples with known concentrations of each analyte was included in eachPCR plate as well as positive and negative controls for each gene. Testsamples were identified by scanning the 2D barcode on the matrix tubescontaining the nucleic acids. Delta Ct (dCT) was calculated from the Ctvalue derived from quantitative PCR (qPCR) amplification for eachanalyte subtracted by the Ct value of actin for each individualpatient's blood sample. Relative expression of patient specimens iscalculated using a standard curve of delta Cts of serial dilutions ofUniversal Human Reference RNA set at a gene expression value of 10 (whenthe delta CTs were plotted against the log concentration of eachanalyte). Preferably, such measurement of RNA expression level can benormalized with one or more expression level of a housekeeping gene,which is generally known in the art not to be affected or notsubstantially affected by abnormalities of the central nervous system,including neural tumors. Any suitable housekeeping genes arecontemplated, and includes, for example, genes encoding cytoskeletalmolecule (e.g., β-actin, etc.), ubiquitin-related protein (e.g.,UBE2D2), a mitochondrial protein (e.g., CYC1, etc.), and/or a ribosomalprotein (e.g., RPL13, etc.).

Without wishing to be bound to any specific theory, the inventorscontemplate that the development and prognosis of the neural tumor isgenerally accompanied with one or more changes, preferably at least twochanges in cfDNA and/or cfRNA detectable in the bodily fluid of thecancer patient. Preferably, such changes include increase and/ordecrease of expression levels of one or more tumor-specific genes, ortumor-specific changes of one or more tumor-related genes. Such findingis particularly unexpected as these tumors are typically insulated fromthe circulation by the blood-brain barrier that is thought to be asignificant obstacle in translocation of molecules the size of cfRNA.Nevertheless, various cfRNA have been detected in patient sera at arelatively large dynamic range with high sensitivity and specificity.

Indeed, there are many suitable cfRNA markers contemplated formonitoring various neural tumors (especially glioma, glioblastoma, andneuroblastoma), and all known RNA sequences and portions thereof thatare associated with neural tumors and cancer in general are deemedappropriate cfRNA markers for use herein. Preferably, such cfRNA markersare tumor type specific. For example, suitable markers for gliomainclude MGMT, 1p/19q, IDH1, EGFR, p53, PI3K, Rb, and RAF, and any knownor idiosyncratic mutant form thereof. Likewise, suitable markers gliomastem cells include CD133, Npm1 (nucleiophosmin/B23), CD15, A2B5, nestin,and ALDH1, and any known or idiosyncratic mutant form thereof. Furtherpotential markers as described in Nature Genetics 49, 789-794 (2017).Additionally, or alternatively, contemplated glioblastoma markersinclude ELTD-1, VEGF, PTEN, EGFR, MGMT, IDH1, cytochrome c oxidase, andany known or idiosyncratic mutant form thereof. Additionally, oralternatively, contemplated markers for neuroblastoma include MYCN,CD44, TrkA, LDH, and NSE, and any known or idiosyncratic mutant formthereof.

Further contemplated markers may include those derived from genesrelated to an immune status of a tumor or a patient, or DNA repairstatus of the tumor cells. For example, cfRNA molecules with particularrelevance to cancer immune therapy, include genes encoding at least aportion of PD-L1, TGF-beta, IL-8, and various other cytokines andchemokines. Therefore, suitable markers also include those that provideinformation on the immune status (e.g., suppressed, subject tocheckpoint inhibition, inflammation, etc.) of a tumor or a patienthaving the tumor. For example, suitable markers related to immune statusmay include those associated with checkpoint inhibition, and especiallyTIM3, LAG3, TDO, and PD-L1, alone or in any reasonable combinationthereof (e.g., PD-L1 with one or more of TIM3, LAG3, IDO, and TDO).

Still further contemplated markers may include those are associated withDNA repair status. Therefore, contemplated cfRNAs include those encodinggenes associated with base excision repair (e.g., DNA glycosylase, APE1,XRCC1, PNKP, Tdp1, APTX, DNA polymerase β, FEN1, DNA polymerase δ or ε,PCNA-RFC, PARP), mismatch repair (e.g., MutSα (MSH2-MSH6), MutSβ(MSH2-MSH3), MutLα (MLH1-PMS2), MutLβ (MLH1-PMS2), MutLγ (MLH1-MLH3),Exo1, PCNA-RFC), nucleotide excision repair (e.g., XPC-Rad23B-CEN2,UV-DDB (DDB1-XPE), CSA, CSB, TFIIH, XPB, XPD, XPA, RPA, XPG, ERCC1-XPF,DNA polymerase δ or ε), homologous recombination (e.g.,Mre11-Rad50-Nbs1, CtIP, RPA, Rad51, Rad52, BRCA1, BRCA2, Exo1,BLM-TopIIIα, GEN1-Yen1, Slx1-Slx4, Mus81/Eme1), or non-homologousend-joining (e.g., Ku70-Ku80, DNA-PKc, XRCC4-DNA ligase IV, XLF).

Additional markers may also include various cancer associated markerssuch as CEA, AFP, and various mutated forms of ras, p53, and/or patient-and tumor-specific mutations that can be identified by comparing omicsdata for the tumor tissue and corresponding healthy tissue of the samepatient. It should be noted that these patient- and tumor-specificmutations may give rise to patient and tumor specific neoepitopes, ormay simply be used as patient and tumor specific tracking signals.

In further contemplated aspects, all contemplated cfRNA markers can beassociated with or otherwise standardized against a housekeeping orreference gene such as β-actin cfRNA (e.g., per ml of plasma) that canserve as a proxy measure for total cfRNA in patients. Other suitableexemplary housekeeping genes include HMGB1, beta-2-microglobulin,HSP90AB1, genes encoding tRNA synthetases, genes encoding histones,metabolic genes such as phosphoglycerate kinase, enolase phosphatase,lactate dehydrogenase, etc. Still further housekeeping genes arepublished elsewhere (e.g., Trends in Genetics (2013), Vol. 29, No. 10,pp 569-574). Alternatively, specific ratios of selected markers may beestablished to monitor treatment with a particular drug (e.g.,neoepitope marker versus immune status marker), or to monitor status ofthe tumor (e.g., glioma specific marker versus apoptosis or necrosismarker). Of course, it should also be noted that the cfRNA marker may bea full-length cfRNA or only a portion thereof.

It is contemplated that the types and numbers of cell free DNA and/orRNA showing changes, and types of changes may vary depending on thetype, status (e.g., prognosis, severity, symptoms, etc.) of cancer, andthe condition of a patient suffering from the neural cancer (e.g.,pre-disposed health conditions, current health conditions, age, sex,geographical area, etc.). Thus, it should be appreciated that one ormore desired nucleic acids may be selected for a neural cancer, diseasestage of a neural cancer, for monitoring of a specific treatment, foranalysis of tumor clonality, presence of a specific mutation, and/oreven on the basis of personal mutational profiles or presence ofexpressed neoepitopes. Alternatively, where discovery or scanning fornew mutations or changes in expression of a particular gene is desired,real time quantitative PCR may be replaced by RNAseq to so cover atleast part of a patient transcriptome. Moreover, it should beappreciated that contemplated analyses can be performed static, or overa time course with repeated sampling to obtain a dynamic picture withoutthe need for biopsy of the tumor or a metastasis.

For example, a neural tumor can be associated with a mutation in gene A,which results in decrease of RNA expression of gene A. In such case, itis contemplated that the cfRNA obtained from patient's sera may show amutation (e.g., a point mutation, a deletion, an addition, etc.) anddecreased cfRNA quantity compared to a healthy individual with a similarphysical conditions (e.g., age, race, ethnicity, health conditions otherthan cancer, etc.). It is also contemplated that cfDNA obtained frompatient's sera may show a mutation (e.g., a point mutation, a deletion,an addition, etc.), yet cfRNA derived from the same gene may not show amutation if the mutation is located in non-coding area of the gene. Insuch case, cfRNA may only show decreased cfRNA quantity compared to ahealthy individual.

In some embodiments, initiation and development of a neural tumor and/ordevelopment of a symptom or neural tumor can be accompanied with achange in sequence (e.g., mutation) and/or an expression level of acfRNA. In such embodiments, the plurality of changes of the cell freenucleic acid can include at least one nucleic acid sequence changes(e.g., mutation, insertion, deletion, etc.) in a cfDNA or cfRNA, and atleast one expression level changes in a cfRNA. It is contemplated thatthe cfDNA or cfRNA having nucleic acid sequence change(s) may be derivedfrom same gene with the cfRNA showing expression level changes. Forexample, the patient's blood may include cfDNA that has one missensemutation of gene A, and cfRNA of gene A that shows decreased expressionlevel in the patient's blood. It is contemplated that the cfDNA with amutation and the cfRNA may be derived from the same portion of the geneA or different portion of the gene A, considering relatively short sizeof the cell free nucleic acid detected in the blood. In otherembodiments, the cfDNA or cfRNA having nucleic acid sequence change(s)may be derived from different gene than the cfRNA showing expressionlevel changes. In such embodiments, it is contemplated that the genes ofcfDNA or cfRNA may be functionally related. For example, the patient'sblood may include cfDNA that has one missense mutation of gene A,resulting in encoding hypo-phosphorylated protein A, and cfRNA of gene Bshows increased expression due to decreased transcriptional inhibitionby hypo-phosphorylated protein A. In another example, the patient'sblood may include cfDNA that has one missense mutation of gene A,resulting in encoding hypo-phosphorylated protein A, and cfRNA of gene Bshows increased expression due to the increased inflammation response byinactive hypo-phosphorylated protein A.

It is also contemplated that the plurality of changes of the cell freenucleic acids can include expression levels of two or more cfRNAs. Insome embodiments, the plurality of cfRNAs that has increased ordecreased expression levels may be derived from same gene. For example,two cfRNAs are two splicing variants of gene A, and only one cfRNAs mayshow increased expression level while another cfRNA has static, or evendecreased expression level. In other embodiments, the plurality ofcfRNAs that has increased or decreased expression levels may be derivedfrom different, distinct genes. In such embodiments, the plurality ofcfRNAs that has increased or decreased expression levels may be derivedfrom genes encoding functionally related proteins. For example, twocfRNAs are derived from genes in the same signaling pathway such thatone cfRNA is derived from gene A and another cfRNA is derived from geneB, where protein A′ encoded by gene A is inhibitory to a signalingpathway that leads to transcription of gene B. For other example, onecfRNA is derived from gene A and another cfRNA is derived from gene B,where protein A′ encoded by gene A stabilize the mRNA encoded by gene B.

Alternatively, the plurality of cfRNAs that has increased or decreasedexpression levels may be derived from genes encoding functionallyrelevant proteins, yet not necessarily located in the same pathway. Forexample, one cfRNA is derived from gene C and another cfRNA is derivedfrom gene D, where gene C is involved in the apoptosis pathway and geneD is involved in the inflammatory response pathway. In such example,those two cfRNA expression levels are not inter-regulated directly, yetmay concurrently be increased or decreased due to the physiologicalconditions of the cell.

In addition, the plurality of cfRNAs and/or cfDNAs that show changes canbe derived from a same type of cells or different types of cells in theneural tumor. For example, cfRNAs and/or cfDNAs that are changed can bederived from neural tumor cells, immune cells in the neural tumor mass,or any other types of cell that interact or surround the neural tumorcells.

It is contemplated that at least some of such changes can be anindicator of a prognosis of the neural cancer when consideredindividually and/or collectively. Typically, such changes arequantitatively and/or qualitatively interrelated with each other, while,alone, may not be a decisive factor for determining a prognosis of theneural cancer. As used herein the term “interrelated” or“interrelationship” refers a relation between changes that are shownamong patients diagnosed with the neural tumor at a statistically higherchance (e.g., at least 50% of the patients, at least 70% of patients,etc.). Thus, it should be appreciated that the interrelated changes maybe causally related (e.g., change A causes change B, etc.) or associated(e.g., change A and change B occurs concurrently without necessarilyhaving a direct or indirect interaction with each other, etc.). Forexample, a cfRNA derived from a mutated EGFR (e.g., EGFRvIII) gene and acfRNA derived from a mutated IDH-1 gene can be detected from a patient'sblood. A mutation in EGFR may be an indicator of a tumor, but notnecessarily of a neural tumor, as EGFR mutations are found in multipletypes of tumors (e.g., non-small cell lung cancer, etc.). Similarly, amutation in IDH-1 may be an indicator of a tumor, but not necessarily ofa neural tumor, as IDH-1 mutations are found in multiple types of tumors(e.g., gliomas, glioblastoma, acute myeloid leukemia, thyroidcarcinomas, etc.). Yet, the concurrent detection of mutation in EGFR andIDH-1 can be a stronger indicator of neural tumor development.

In another example, from a patient's blood, decreased expression levelof cfRNA derived from Adhesion G Protein-Coupled Receptor L4 (ADGRL4)gene and a specific alternatively spliced form of cfRNA derived fromHomeobox B3 (HOXB3) can be detected. While decreased transcription ofADGRL4 can be often detected in glioblastoma, it may neither be adecisive factor for glioblastoma development as such change can beobserved in different types of tumors. Yet, the concurrent detection ofdecreased transcription of ADGRL4 and specific alternatively splicedform of HOXB3, collectively, can indicate the recurrent glioblastomadevelopment. In such case, it is contemplated that the prognosis ofglioblastoma can be determined based on the magnitude of decreasedexpression of ADGRL4 (e.g., at least 30%, at least 50%, at least 70%, atleast 80% decrease compared to a healthy individual, etc.) and the ratioof the alternatively spliced form of HOXB3 (e.g., at least 30% totalHOXB3 mRNA, at least 50% total HOXB3 mRNA, at least 70% total HOXB3mRNA, etc.).

It is also contemplated that a score can be calculated based on theplurality of changes, which can be used to determine prognosis of theneural cancer. In some embodiments, each change of relevant cfDNA andcfRNA may be given a positive or a negative score to add up to generatean overall score of the patient's cell free nucleic acid. For example,where decreased transcription of ADGRL4 and specific alternativelyspliced form of HOXB are detected, a cfRNA score for ADGRL4 can becalculated based on the magnitude of the ADGRL4 mRNA expression (e.g., 1score per 10% decrease, etc.), and cfRNA score for alternatively splicedform of HOXB can be calculated based on the quantity ratio of thealternatively spliced form of HOXB among overall HOXB mRNA (e.g., 1score per 10% total alternatively spliced form of HOXB among overallHOXB mRNA, etc.). Then, the prognosis of the glioblastoma can bedetermined (e.g., diagnosed, the progress is confirmed, etc.) when theoverall score exceeds a predetermined threshold. In other embodiments,the score can be calculated based on the plurality of changes in asliding scale. For example, where the prognosis of the glioblastoma canbe determined by determining ADGRL4 mRNA expression level and HOXB mRNAalternatively spliced form ratio, the prognosis of the glioblastoma canbe determined when the ADGRL4 mRNA expression level is increased over200%, over 300%, over 400% even if HOXB mRNA alternatively spliced formratio is less than 20%, Alternatively, the prognosis of the glioblastomacan be determined when the HOXB mRNA alternatively spliced form ratio ismore than 70%, more than 80%, more than 90%, even if the ADGRL4 mRNAexpression level is increased less than 30%, less than 20%, or less than10%.

Additionally, the prognosis of a neural tumor can be determined bydetecting changes of relevant cfDNA(s) and/or cfRNA(s) over time thatreflect relative changes of cfDNA(s) and/or cfRNA(s) in the patient. Forexample, changes of cfDNA(s) and/or cfRNA(s) can be detected in thefirst and second samples of the patient, which are obtained at least 1day, at least 5 days, at least 10 days, at least 30 days, at least 3months, at least 6 months, at least 1 year apart. The time distancebetween two samples may vary depending on the type of neural tumors. Forexample, it is preferred that the time distance is shorter (e.g., 7days, 2 weeks, 1 month, etc.) for a fast-developing neural tumors (e.g.,grade 4 astrocytoma, etc.). For other example, it is preferred that thetime distance is relatively longer (e.g., 1 month, 3 months, 6 months,etc.) for a slow-developing neural tumors (e.g., childhood brain tumors,etc.).

While the sets of cell free nucleic acids that may show changes in thefirst and second sample of the patient may differ depending on the typeor prognosis of neural tumors, it is preferred that the sets of cellfree nucleic acids may have at least one cell free nucleic acid incommon for comparison. For example, the set of cell free nucleic acidsin the first sample that show changes may include cfRNAs of gene E andgene F, and the set of cell free nucleic acids in the second sample thatshow changes may include cfRNAs of gene E and gene G.

The inventors also contemplate that the multiple measurements of thecell free nucleic acids over a period of time can be used to determinethe effectiveness or likelihood of successful outcome of the treatmentto the neural tumor(s) and/or to provide a recommendation of treatmentregimen. For example, changed expression levels of cfRNA derived fromgene H and I can be detected before and after a treatment (e.g., a drugtreatment, a radiotherapy, a surgery, etc.), or multiple time pointsbefore and after the treatment to evaluate the overall trend of changesin the expression levels of cfRNA H and I. The cfRNA H and I'sexpression levels are both increased during the multiple time pointsbefore the treatment. After the treatment, decrease of expression levelsof cfRNA derived from gene H could be detected, while the expressionlevel of cfRNA I is relatively static. In such example, a treatment canbe considered to be effective to stop or reverse the progress of theneural tumors (as reflected by decrease of cfRNA H). In addition, atreatment regime to affect a pathway involving gene I (e.g., a drugtargeting the protein encoded by gene I, a drug targeting thetranscription pathway of gene I, a drug targeting a protein upstream ordownstream of the protein encoded by gene I, etc.) can be recommendedfor the next treatment plan or can be administered to the patient totreat the tumor based on the determined prognosis of the tumor with theexpression levels of cfRNA H and I or other cell free nucleic acid.

Alternatively and/or additionally, measurement of cfDNA and/or cfRNA canbe analyzed in view of one or more a behavioral test data (e.g., motorbehavior, sensory perception, etc.), a cognitive test data (e.g.,psychometric assessment, etc.), electroencephalography (EEG) data,electromyography (EMG) data, and a MRI and/or CAT scan data. The typeand combination of the test data may vary depending on the type of theneural cancer, the suspected location, and/or progress of the neuralcancer. For example, for patients suspected to have a neural cancer in asize large enough to be detected as a tumor mass, a CAT scan data and/ora MRI test data may be accompanied or used as supplemental to detectionof changes in cfDNA and/or cfRNA. In another example, the functionaleffect of the neural cancer can be determined by behavioral test data(e.g., neural tumor in cerebellum may be accompanied with the motorbehavior disability including loss of balance, etc.) or cognitive testdata (e.g., for neural tumors located in the frontal lobe or temporallobe, etc.).

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

As used herein, the phrase “at least one of A and B” is intended torefer to ‘A’ and/or ‘B’, regardless of the nature of ‘A’ and ‘B’. Forexample, in some embodiments, ‘A’ may be single distinct species, whilein other embodiments ‘A’ may represent a single species within a genusthat is denoted ‘A’. Likewise, in some embodiments, ‘B’ may be singledistinct species, while in other embodiments ‘B’ may represent a singlespecies within a genus that is denoted ‘B’.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise. Unless the context dictates the contrary,all ranges set forth herein should be interpreted as being inclusive oftheir endpoints, and open-ended ranges should be interpreted to includecommercially practical values. Similarly, all lists of values should beconsidered as inclusive of intermediate values unless the contextindicates the contrary.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

1. A method of monitoring status or treatment of a neural cancer of apatient, comprising: obtaining a sample of a bodily fluid of thepatient; and measuring at least one cfRNA in the bodily fluid of thepatient, wherein the cfRNA is specific to at least one of the neuralcancer, a DNA repair status, and an immune status.
 2. The method ofclaim 1, wherein the bodily fluid is whole blood, plasma, or serum.3-13. (canceled)
 14. The method of claim 1, wherein the neural cancer isa glioma, a glioblastoma, or a neuroblastoma.
 15. The method of claim 1,wherein the cfRNA is a cfRNA from a neural tumor cell and is selectedfrom the group consisting of MGMT, IDH1, EGFR, p53, PI3K, Rb, RAF,CD133, CD15, A2B5, nestin, ALDH1, ELTD-1, VEGF, PTEN, cytochrome coxidase, MYCN, CD44, TrkA, LDH, NSE, and any mutant form thereof. 16.The method of claim 1, wherein the cfRNA is a cfRNA associated with DNArepair selected from the group consisting of base excision repair,mismatch repair, nucleotide excision repair, homologous recombination,and non-homologous end-joining.
 17. (canceled)
 18. The method of claim1, wherein the cfRNA is a cfRNA associated with an immune status. 19.The method of claim 18, wherein the cfRNA encodes at least a portion ofa gene encoding at least one of PD-L1, TIM3, LAG3, IDO, and TDO.
 20. Themethod of claim 1, further comprising a step of measuring at least oneadditional cfRNA in the bodily fluid of the patient, wherein the atleast one additional cfRNA encodes β actin, HMGB1, β-2-microglobulin, orHSP90AB1.
 21. (canceled)
 22. (canceled)
 23. The method of claim 1,further comprising a step of calculating a ratio of a first cfRNA and asecond cfRNA in the bodily fluid.
 24. The method of claim 1, furthercomprising a step of modulating a barrier property of the blood-brainbarrier in the patient.
 25. A method of determining a prognosis of aneural cancer of a patient, comprising: obtaining a first sample of abodily fluid of the patient; detecting a plurality of changes of one ormore cfRNA in the first sample, wherein the plurality of changes arerelated to a prognosis of the neural cancer, wherein the cfRNA isspecific to at least one of the neural cancer, a DNA repair status, andan immune status; and determining the prognosis of the neural cancerbased on at least one of an interrelationship among the plurality ofchanges and a predetermined threshold of at least one of the pluralityof changes.
 26. The method of claim 26, wherein the bodily fluid is atleast one of whole blood, plasma, serum, and cerebrospinal fluid. 27-42.(canceled)
 43. The method of claim 25, wherein the plurality of changesis selected from a group consisting of a mutation, differentialexpression of splicing variants, an overexpression, an underexpression,a maturation.
 44. The method of claim 25, wherein the plurality ofchanges comprises a mutation in a first cfRNA and an overexpression of asecond cfRNA, wherein the first and second cfRNAs are derived from twodistinct genes.
 45. (canceled)
 46. The method of claim 25, wherein theplurality of changes comprises expression levels of a first cfRNA and asecond cfRNA, wherein the first and second cfRNAs are derived from twodistinct genes in a same signaling pathway. 47-49. (canceled)
 50. Themethod of claim 25, wherein the interrelationship among the plurality ofchanges is measured in a sliding scale.
 51. The method of claim 25,comprising calculating a score for the plurality of changes, andcomparing the score with the predetermined threshold.
 52. The method ofclaim 25, further comprising modulating a barrier property of theblood-brain barrier in the patient.
 53. The method of claim 25, furthercomprising: obtaining a second sample of a bodily fluid of the patientin a different time point than the first sample, wherein the differenttime point is a post-treatment time point, and further comprisingdetermining an effectiveness of a treatment based on the plurality ofchanges of the first and second sample; detecting a plurality of changesof the one or more cfRNAs in the second sample, wherein the plurality ofchanges of the first and second sample include changes of at least onecommon cfRNA; and determining the status of a neural cancer by comparingthe plurality of changes of the first and second samples.
 54. (canceled)55. (canceled)
 56. The method of claim 25, the determining the prognosisof the neural cancer is further based on at least one of a behavioraltest data, a cognitive test data, electroencephalography data, and a CATscan data.
 57. (canceled)
 58. (canceled)